The cellular control mechanisms involved in the stimulus-secretion coupling mechanism of rat submandibular cells were studied using a dispersed cell suspension technique developed in this laboratory (Proc. Natl. Acad. Sci. (USA) 76: 2789-2793, 1979). The secretory response was evaluated by determining the amount of C14-glucosamine-labeled mucins released in a 40 min period. The cellular levels of cAMP were determined using a RIA assay following deproteinization, purification via column chromatography, and acetylation. The removal of calcium from the incubation medium did not prevent the secretion of C14-labeled mucins nor did it prevent the increase in cellular levels of cAMP following beta-adrenergic receptor activation. Prior incubation of the cells in calcium-free medium containing 5 mM EGTA prevented the secretion of C14-glucosamine-labeled mucins following beta-adrenergic stimulation but it did not prevent the cellular increase of cAMP. Cyclic AMP analogs were able to elicit a secretory response in the absence of beta-adrenergic receptor activation but the analogs were unable to reverse the inhibitiory effect of prior incubation in 5 mM EGTA. These data suggest that cellular calcium as well as increased cellular levels of cAMP mediate the secretion of mucins from these cells.